JPH06511018A - Plasma fractionation purification method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Plasma fractionation purification method field of invention The present invention provides purified factor X6, phospholipids, Ca'', and factor V.

Purified using plasma fractions containing Prothrombin (factor 1) to thrombin (t A method useful for preparing factors ([1, V, and and X,) resulting from a single purification procedure.

Background of the invention The initiation of blood clotting involves two different, but similar, intrinsic and extrinsic coagulation pathways or It is caused by a molecular mechanism called a cascade. This endogenous coagulation pathway It also contains tissue factor. Each of the reaction steps of these two cascades Well, Brotainase...

It is converting an inactive zymogen into its enzymatically active form. of that cascade The last step is the same for both the intrinsic and extrinsic routes, but , inactive prothrombin is converted to thrombin, which in turn is converted into soluble filament. Catalyzes the conversion of brinogen to insoluble fibrin.

The conversion of zymogens to active blotinase in both cascades is dependent on cofactors. Very slow in non-existence. This cofactor is observed in their absence. Stimulates zymogen activation rates from 10' to 105 times the observed rate. child In the cascade of (3) acidic phospholipids derived from the membrane bilayer of the plate or damaged tissue; protein cofactors that are specifically required for the activation of the protein. Approximately 69,000~ Prothrombin, which has a molecular weight of approximately 80,000, consists of one polypeptide chain. Ru. Similar to the activation of all blood coagulation zymogens, activation of prothrombin is , negatively charged phospholipids derived from platelets or damaged tissue, e.g. Such lipids occur on the surface provided by dilserine, which contributes to the lipid division of cell membranes. The stratified layer is located mostly on the cytoplasmic side or inside. Prothrombin is If the cysts are destroyed and do not expose their inner surface, healing may occur in the red blood cells or endothelial cells. I don't wear it. Therefore, cell destruction that exposes negatively charged phospholipids is a process that )・Enables Longin to bind to the above phospholipids. This is the It is the first step in the activation of rotombin. The above phospholipid of prothrombin Binding to protein is a Ca2''-dependent process.

Activation of prothrombin also binds to phospholipids through Ca”-dependent interactions. is catalyzed by factor X. However, the maximum activation of prothrombin The large rate is factor V, also the above prothrombin-X, -Ca''-phospholipid complex Obtained only when attached to the body.

Factor X is a glycoprotein with a molecular weight of about 59,000 to about 70,000; two subunits, one subunit with a molecular weight of approximately 40,000. knit and about 19,000 other subunits. Factor X is an endogenous is activated by factor X, both in the tract and extrinsic pathways.

Factor ■, which has a molecular weight of 330,000, is one of the essential factors for the activation of ■. It is a glycoprotein consisting of one polypeptide chain containing tightly bound Ca. 1 is not a proteinase, but serves as an auxiliary protein for prothrombin activation. This acts to increase the rate of prothrombin activation. Factor V itself is activated by thrombin and is therefore involved in the thrombin activation reaction. The generated thrombin or factor ■ is activated to factor ■, which in turn Increases hin synthesis speed.

In vitro studies have shown that Ca' and phospholipids are significantly faster at conversion rates than those of Factor X alone. The conversion rate of prothrombin to thrombin by factor It was shown that the degree of The factor ■, is about 350 times the conversion speed by increase, but once the factors are together, it will be at least about 20,000 times faster. Increased from time to time. Thus, the Ca''-phospholipid-X, -V complex was within 1 minute to produce the same amount of thrombin as would be produced by Factor X alone. to create.

Thrombin converts fibrinogen into fibrin, the substance that forms blood clots. Being an active agent in the conversion to soluble proteins, thrombin s), from bleeding ulcers, surgical procedures, and other such injuries. has uses in the prevention of blood loss. Natural blood clotting allows blood to flow from the injured area. It takes several minutes to effectively stop the flow of water. However, damage The use of thrombin at the site provides immediate coagulation. Therefore, Trombi Typical uses for this drug include treating symptoms that exist after surgery and trauma. in the treatment of ulcers to prevent excessive blood loss, which is preferable or fatal. Preferred for oral use. Also, thrombin into the vitreous cavity during eye surgery Injections are difficult to identify for cauterization. Bleeding from difficult areas can be prevented.

Such treatments are used to treat symptoms caused by increases in intraocular pressure during routine surgical procedures. reduce the risk of eye damage.

Recently, bovine thrombin preparations are available for current use. but However, bolus thrombin preparations often have disadvantages within the patients being treated. elicit the desired immune response. Such a response may be due to the use of human thrombin preparations. This can be avoided by However, human thrombin preparations are expensive and , thus reducing the need for their use. Therefore, is it a human source? et al., the economic effects of purifying prothrombin and activating it to thrombin. There is a need for certain measures.

Summary of the invention The present invention uses Factor V and Factor X to activate Factor 11 to Factor II. 1, wherein factors [1, ■, and X are derived from a single impure protein fraction It describes the method by which it can be obtained.

This activation method involves adding factor 11 to the activation buffer and adding factor 11 to the activation buffer. [Provide solution It consists of the process of This factor]I is added to an aqueous solution of an impure protein fraction. Prepare by Factors II, ■ and X contained in this impure protein fraction is bound to a DEAE ligand and then recovered from the DEAE ligand. do. The protein fractions of the recovered factors] and X were precipitated by adding barium chloride. Make me proud. This barium chloride precipitate is dissolved and then bound to factor child (1 is bound by a ligand that binds weakly or in some cases not at all) Applicable to chromatography resins. factor I], its factor Moth: It is recovered from both parts of the moth without binding or weakly binding to the node.

Factor V is added to the Factor 1 solution described above to provide a Factor 11/Factor V solution. child Here, this factor recovered from the barium chloride supernatant. factor V, factor ++, factor] Activates during sexualization reaction. This activation reaction (here, factor +1 is factor ■It is activated very quickly in the absence of the factor. However, a small amount of the factor II, is created.

These small amounts of factor I+ are then obtained and factor ■ is activated to factor ■8. and several factors are produced during this purification process. like this Factor V is then obtained and allowed to interact in the factor II activation reaction described above. can be done. This is due to factor I1. resulting in an increase in the rate of synthesis. that Therefore, a separate activation step for factor ■ is not necessary.

Factor X is added to the above factor [1/factor ■ solution, and factor [1/factor , provide a solution. This factor X is used as the factor It is prepared by recovering factor X from the sample.

Phospholipid membranes and calcium ions are added to this factor [1/factor ■/factor and the resulting mixture was mixed with Factor I] and Factor I1. to activate Incubate.

Brief description of the drawing Features, aspects, and advantages of the invention will be apparent in the following detailed description, appended claims, and more. The accompanying figure is a flow diagram illustrating a preferred embodiment of the method of the invention. It will be more fully understood when considered in terms of aspects.

detailed description The method provided in accordance with the practice of the present invention is based on the method provided in accordance with the practice of the present invention. , ■ and X. As used herein, "impure protein fraction" (impure protein fraction) + is factor 11, ■ and This means that it contains one or more proteins in addition to X. Factor X is then activated, and Added to activation reactants that also contain calcium, phospholipids and factor 2. , which activates its factor II zymogen into the active catalytic factor [1, .

Purification of factors I+, ■ and X The purification of factors 11, From human plasma collected and tested according to procedures approved by the Drug Administration. It is. The plasma is optionally cryoprecipitated prior to this purification step. Can be done. This plasma fraction is electrodialyzed to determine its sodium concentration from its original value. It can be reduced to between 85 and 105mM. This dialyzed plasma is Next, the pH is adjusted to neutrality by adding acetic acid.

Factor II V, [X and X contained in this pH-adjusted plasma are (diethyl aminoethyl) cellulose, DEAE Sephadex, or other suitable a suitable ion-exchange medium, i.e. a buffer, e.g. at a pH of about 6.8. about 0.03M sodium phosphate and about 0.03M sodium citrate (phosphorus). acid/citrate buffer) and then the factors [l, Adsorb onto a matching medium. This DEAE resin and cryoprecipitated plasma were Mix for 0 minutes and the DEAE resin is then collected by centrifugation. This D The EAE resin is washed with a buffer, such as a phosphate/citrate buffer.

Discard this cleaning solution. This washed DEAE resin is washed with a buffer, e.g. Suspend in citrate/citrate buffer. The resulting suspension is then rinsed into the column. and discard the eluate. This DEAE resin can be mixed with a buffer, e.g. Wash with acid/citrate buffer and this wash is also discarded. factor It, V,] IX and X in about 0.03 M sodium phosphate at a pH of about 6.8. about 0.0381 thorium citrate containing about 0.2 kl NaCl The DEAE resin is washed by elution with a buffer comprising This allows for elution. This eluate was collected and factor Il5V, IX and X-containing fractions are pooled and collected into bulk solution. This recovered Perform appropriate testing of the fractions and adjust the pH of this bulk solution to near neutrality. This solution is then filtered through a sterile Hactia-retaining cartridge or membrane. and thereby create a bulk solution of Factors II, V, IX and X. This bulk solution Freeze until further processing.

For further processing, thaw this bulk solution and millipore Provided by Carp, of Bedford, MA”^l1llip Perform diafiltration using “ore Pelicon” concentrator. , the eluate can be concentrated to about 10 g protein/l. one exemplary situation In this case, a bulk solution of factors 11, V, IX and Inactivates any virus contaminants contained in the virus. Inactivate this virus Step (solvent-detergent treatment or D- This diafiltration bulk solution is treated with v/v)% Tri-(n)butyl phosphate and about 10% polysorbate. 80. Approximately 0.08~0.14k g of Tri-(n)butyl phosphate, polysorbate 80 solution in bulk. Add per kg of solution. The resulting solution was heated at about 27°C to about 6 to about 8 time, mix. The treated factor ll-1V-1IX- and χ-containing solution was further processed according to the process of the present invention.

Purification of Factors I, ■ and X by Barium/Citrate Precipitation One of the Practices of the Invention In an exemplary embodiment, a virus inactivating agent made as described above [[-, V-1[X-, and 0.02 M sodium citrate at a pH of 7.5 approximately 2-10 times Dilute. If desired, a solution containing factors +1-, V-1IX-, and virus has been inactivated, or by a method other than those described above. Those in which viruses have been inactivated can be used.

0.5-2M barium chloride solution sufficient to precipitate factors]], IX, and X. to the above diluted 11-, V-1IX-, and X-containing solutions. Ru. The barium chloride concentration obtained is about 0.1 to about 0.2M. This precipitate ( Barium or Ba precipitate) is recovered and 0.2 to 0.6 M Dissolve in EDTA ((ethylene-dinitrilo)tetraacetic acid) in solution, and As previously described, a low-sodium solution was used to remove barium and EDTA. 0 to about 0 in a solution buffered to a pl of between about 6.0 and about 9.0 Perform diafiltration on 0.2M NaC1) and further Obtaining a favorable low sodium concentration for processing. This diafiltration Suitable buffers for this purpose have a buffer of about 0.02λ1 at a pH of about 6.6 to about 7. and about 105 M NaCl.

The supernatant containing factor ① was mixed with approximately 0.06 M Tris, as described above. pH7,2,0,09! , I NaCl, and 0.01 M CaCl2 A separate diafiltration is performed on the containing buffer. factor Freeze the V-containing solution until needed for use.

Barium chloride precipitation reduces the specific activities of factors 11, IX and X from about 1.2 to about 3. This led to an increase in

This diafiltered and dissolved barium precipitate was extracted with dextrose sulfate. Applied to a column containing silica resin combined with Tolan (DSS).

Preparation of dextran sulfate-silica resin In one exemplary embodiment of the preparation of SS resins useful in practicing the principles of the present invention, Under the registered trademark CNBR5P500, 5ERVA Fine Bioch Supplied by electronics Inc., of Westbury, NY , activated silica resins as described in U.S. Pat. No. 4,725,673. and is incorporated herein by reference. Simply put, that Ikg of resin, supplied in CNBr-activated form, was mixed with 2kg of distilled water. and then slurry gently in a Buchner funnel. ness). This slurry/wash step was repeated two more times. sulfuric acid Add the dextran solution to a buffer solution, preferably about 0.0%. IM NaHCO Dissolve 10 to 2 oo grams of dextran sulfate in 3 ml (at a pH of about 8.3). prepared by decomposition and the washed silica resin and dextran sulfate Combine the solutions and mix for up to about 24 hours to remove the dextran sulfate. - Attach the ligand to its activated silica resin. This ligand-binding resin is Rinse three times with at least 1 volume of distilled water in a Puchner funnel and aspirate from the moisture reservoir. did. This ligand-conjugated resin is then further added using a Buchner funnel to approximately 6 to At a pH of 8, about 2.0 to about 4. Wash several times with a solution containing OM NaCl. Clean and aspirate into a humidifier. This resin is further added to approximately 6 to 8 volumes of 1 volume or more. A similar solution with a buffer solution containing about 0 to about 0.2 M NaCl at pH Cleaned by washing and suction.

This washed ligand-binding resin was then mixed with several volumes of serum albumin, Treat by mixing for up to 0 hours to remove the "tree-" binding groups. Blocked both. This blocked ligand-binding resin was The tube was washed several times with distilled water to remove excess blocking agent. This block 2.0 to about 4.0, at a pH of about 6 to 8. O Washed several times with one or more volumes of a solution containing M NaCl. Final wash with distilled water was provided and the resin was drawn into a humidifier.

The final step in preparing the ligand-binding resin reservoir is to add several resin volumes of the “alkatone” (alkatone) catone)” (50% by volume acetone, 35% by volume ethanol, and 15% by volume) % water solution). This slurry is used for It was previously stored at 2°C to 8°C. This resin is decomposed from the above alkatone. Cantate and wash with distilled water at a pH of approximately 6.8 and pack into the column. and about 0 and about 0.05 M at a pH of about 6 to about 9 prior to use. Equilibrated with NaCl between Equilibrate this chromatography resin A suitable buffer is about 0.02 M citric acid at a pH of about 6.6 to about 7. and about 0.05M NaCl.

Factor] Separation of factor 1 and factor Usually between 2 and 5 liters of dextran sulfate silica resin is processed. virus inactivation factor IL [required for 5 kg of each of X and X solution] It is. This dextran sulfate-silica resin is preferred. If so, this series The resin is easily used in column, chroma dog, and roughy procedures. Because it is not compressed and therefore maintains the desired flow rate throughout the stages as above It is.

However, other resins, such as Sepharose, could also be used. Dew. Also, although dextran sulfate is specifically mentioned, other adhesives, such as heparin Phosphorus could also be used.

The solution containing the diafiltered barium precipitate was prepared as previously described. Prepare sea urchin. This solution is then mixed with the factor contained in the solution [X and The dextran sulfate is passed through the silica resin in the column so that it is absorbed onto the resin. Then I sent him a pump. Factor] 1 remains unadsorbed or is slowly adsorbed. and in the breakthrough eluate or its It is washed from the column early in the later elution step. The end of this adsorption stage After that, the Factor IX-, Wash with buffer volume. Suitable wash buffers have a pH of about 6.6 to about 7. and contains about 0.02M sodium citrate and about 0.05M NaCl. It consists of A2 that goes beyond the background. . with the eluate and wash solution (i.e., A2g obtained for the buffer eluted from the column during the equilibration step . reading), both of which contain factor ]I, or pool them. This factor II-containing fraction Either immediately further process or freeze and store for subsequent processing. or one of the following.

Factors (X and about 0.05M to about 0.6M NaC in a buffer solution comprising thorium The resin is eluted with a linear salt (NaCl) gradient up to l. This N Factor X elutes from the column when the aC1 concentration is approximately 0.2+11. factor X-containing fractions are pooled and processed immediately or for subsequent processing. Either is frozen and stored. Optionally, factor X-containing fraction were reapplied to the DO3 resin and for further purification as described above. can be eluted. This Factor X eluate was collected prior to further processing or freezing. can be filtered. Separately pooled factors from several operations +1, [ When the X and X fractions have been collected, they are each further processed.

When the above salt gradient reaches a concentration of about 0.25M to about 0.4&l; Both +X and X elute. At concentrations above about 0.11, Factor IX Only eluted. Pool fractions containing Factor IX and immediately further process. or frozen and stored for further processing.

For further processing, the factor X-containing fraction is thawed (if frozen) and and the pH is adjusted to approximately neutral.

This combined pool is then placed in an activation buffer, e.g., at a pH of about 7.4. about 0.02 M HEPES (4-(2-hydroxyethyl)-1-pipera) Diafifi Accurate target parameters of Factor X activity and sodium concentration - get. This is preferably 0.029 I HEPES, pH 7°4 and 0.15M NaCl.

A sterile bulk of Factor X is sampled for Factor X activity.

This Factor X can be frozen until there is a demand for use.

For further processing, the factor II-containing fraction is thawed (if frozen) and and adjust the pH to approximately neutral.

This combined pool was then ultrafiltered to concentrate Factor II, and Factor 1[ Obtain accurate target parameters of activity and sodium concentration.

Check and readjust the pH if necessary. If desired, factor I Other methods of concentration of I, such as ammonium sulfate precipitation, etc.

diafiltration after diafiltration, or other suitable means known to those skilled in the art. can be used.

Factor I+ can be frozen or lyophilized until required for use. Ru.

Activation of factor X into factor In the blood, factor X is linked to factor Vlll in the endogenous cascade.

, by factor IX in the presence of calcium ions and phospholipids, and exogenously In the pathway, in the presence of factor I11, phospholipids, and calcium ions, It is activated by factor Vll. In the present invention, activation of factor This can be achieved through the action of these blood factors. Alternatively, other proteolytic enzymes For example, Russell's viper venom. nom) can be used.

Preferably, Factor X is purified by the procedure described above.

Snake venom proteins obtained from Vipera russelli snake venom Incubation with white-degrading enzyme activates its active catalytic form. sexualize.

Approximately 2 g of Factor Incubate with 005M CaClt for 30 minutes at 37°C, Convert factor X to factor X. At the end of this activation, the snake venom proteolytic enzyme benzamidine-sepharose (Pharmacia of Uppsa) la, Sweden) for column chromatography. Remove. This benzamidine-Sepharose is added to an activation buffer, e.g. For example, 0.02 M HEPES, pH 7.4, and 0.15^I NaCl Equilibrate by The activator X is applied and the benzamidine- Connect to the seperate interface. Activate factor X.

Chromatography medium containing approximately 0.005 M benzamidine in the sofa Wash it. The eluted factor CI, pH 5.5, and diafiltration for NaCl. and 0.1 (wt/wt)% albumin and I (wt/wt)% It can be stabilized by adding polyethylene glycol or glycerol. Ru.

Activation of factor 11 to factor I+, Factor [l factor 11. activation in the presence of phospholipid membranes and calcium. This is achieved by the action of both factor X and factor (2).

Phospholipid membranes are synthesized from individual phospholipids or from phospholipid/fat emulsions, For example, under the trademarks 5OYCAL and FLUO3OL, Alpha Therap eutic Corporation of Los Angels, CA It can be provided by the products sold more. Sonication of phospholipid membranes individual phospholipids, such as phosphatidylserine and phosphatidylcoli. It can be synthesized from

Purified factor ■ is obtained as described above, or alternatively, it is not pure and Unfractionated plasma can be used. Factor ■ is factor I1. factor [1 During the activation procedure, it is activated. In the activation reaction, factor 11 is combined with factor V , is activated very slowly in the absence of . However, a small amount of A child If is created.

These small amounts of factor I1. is used to activate factor ■ into factor ■, and some V is also created during the purification process. Therefore This generated factor V is then allowed to interact in a factor 11 activation reaction. 1, which is the factor I1. Also increases in synthesis rate Tarasu. Therefore, no separate activation step for factor V is necessary.

To activate factor II, factor I+ obtained as described above was added to about 7.3 from about 2 to about 20 A in about 0.06 kl Tris buffer at a pH of Dilute to 280 units and add approximately 0.09 M NaCl, approximately 0.1 μg/ml. factor v fraction, about 5 to about 50 nmol/ml. Phospholipid, approximately 2-20mM CaCl2 (final concentration), approximately 0.3% Tri- (n) butyl phosphate, and about +(wt/wD%) polysorbate 80 (final concentration). This mixture is heated at about 24°C to about 30°C for about 5 to about 6 hours. Incubate for an hour. At the end of the incubation, this mixture The pH of the solution was adjusted to about 6.5 and the solution was enriched with about 5 (wt/wt)% PE. It can be clarified by addition of G (final concentration) and centrifugation.

factor [1, to Pharmacia of Uppsala, Sweden Chromatography on sulfapropyl-Sephadex supplied by is recovered from the supernatant using other suitable chromatography resins.

The chromatography medium is treated with a buffer, e.g., at a pH of about 6.5. Equilibrate with approximately 0.01M sodium citrate. This chromatography medium Quality factor I1. After applying the inclusions, the chromatography medium is about 0.01M sodium citrate or other suitable buffer at a pt+ of 6.5 Wash with air. This factor I1. is a buffer, e.g., at a pH of about 6.7. The chromatography medium is eluted with approximately 0.1 M sodium citrate. put out This factor I1. , and perform diafiltration, Its composition is about 5-1 ontl histidine, pH 6.7, about 0.1-0.2% C a ion, and adjust to about 0.1-0.2M NaCl. This solution then , previously sterilized) using a cutillary retention cartridge or membrane filter. Sterile filtered and lyophilized.

Factor +1. In one embodiment of the practice of the invention for the purification of V, IX and In this case, factors I], V, tX, and X contained in the cryoprecipitated plasma were 0.03M sodium phosphate and 0.03M sodium citrate at a pH of 6.8. Adsorbed onto DEAE cellulose equilibrated with thorium. Second DE AE cellulose and plasma were combined for approximately 30 minutes and collected by centrifugation. DEAE cellulose in 0.03AI sodium phosphate at a pH of 6.8 and 0.03M sodium citrate.

This washing solution was discarded.

This washed DEAE cellulose was washed with 0.03M phosphoric acid at a pH of 6.8. Suspended in 1-lium and 0.03M sodium citrate and in the column I rinsed it. The second eluate was discarded. This DEAE cellulose was brought to a pH of 6.8. Wash with 0.03M sodium phosphate and 0.03M sodium citrate in , and this cleaning solution was also discarded. Factor 11, v, rx and X to 6,8 pl 0.03M sodium phosphate and 0.03M sodium citrate and 0.03M sodium phosphate and 0.03M sodium citrate in The DEAE cellulose was eluted by washing with 2M NaCl. this The eluate was collected and factor [[-, V-1IX- and X-containing fractions were pooled. , and recovered in bulk solution. Apply this solution to a sterile bacteria-retaining cartridge Filtered through and lyophilized. This lyophilized powder was dissolved in heptane. Virus inactivation was achieved by suspension and heating at 60°C for 20 hours. hepta was removed by drying.

! ’] 2.04 kg of dry powder for injection of approximately 64.5 kg Reconstituted with cold water (CWFI). This reconstitution powder was added to 266,6 kg of 0.02 111 Sodium Citrate, pH 7,4, and 0,25 kl at 4°C Diluted with NaCl and incubated for 20 minutes at 2°C to 4°C.

Approximately 53.4 kg of 1.0^1 barium chloride solution (4'C) was added after 2 hours. and the mixture was stirred for an additional hour. a mixture of two , maintained between O″C and 4°C during addition of barium chloride and during mixing. This solution was then spun in a 5 harple S centrifuge at 1 liter per minute. Keeping the flow rate through the centrifuge between 0.2 and 0.6 per Centrifuged. Approximately 10 kg of barium chloride precipitate was thus recovered. factor A supernatant containing ① was collected.

This factor ■ containing supernatant was filtered through MiHipore TP Cartrinon filter Thoroughly scrubbed to remove any particles. This solution was added to Millipore. Pass through the Pelicon concentrator and l/30 and 175 of its original volume The a contraction was between 0 and 0. This concentrated solution was then treated with 0.06M Tris, 0. Diluted 5 times with 09M NaCL, pH 7.2 (-TBS” solution). During the dilution, the material was reconcentrated to its volume before its TBS dilution. This dark The contraction and TBS dilution steps were repeated three more times. Concentrate the factor solution one last time and then diluted to 1/10 to 1/30 of its original volume with TBS. this In this respect, the conductivity of this solution was approximately the same as that of the TBS solution.

About 66,7 kg of 0.4 at 20°C to 25°C to barium chloride precipitate Add 1% IεDTA solution to dissolve the precipitate;

Filter through Millipore TP cartridge filter to remove particles. I left. After filtration, the solution was passed through a Millipore Peliconll condenser. was passed through and concentrated to between 115 and 171O of its original volume. this The concentrated solution was then diluted with 0.02M sodium citrate and 0.05M NaCl diluted to original volume. This concentration and dilution step was repeated 6 or more times. . In this regard, the conductivity of this solution is 0.02M sodium citrate and 0. It was almost the same as that of 505 M NaCl solution. After the final dilution, this die The weight of Affiltration material is 7G. It weighed 4 kg. This redissolved precipitate , factors It, IX, and X.

Half of the diafiltration material containing factors If IX and X (38, 6 kg) at a flow rate of approximately 170 tnl/min, 18 cm y, 94 cm Moduline chromatography column with dextran sulfate silica washing buffer (0.02 kl sodium citrate and and 0.05 M NaCl, pH 6,8). did.

The eluate containing Factor] I was collected. In the end, 425 kg of wash buffer passed through this column.

Approximately 25 liters (1 column volume of wash) of the first wash solution was added to the solution of Factor I [described above]. Combined with exudate. This pool of factor II is &Ii]1ipore Pe1 ! Concentrated by diafiltration using a 1cona condenser.

a diameter reduction, and this factor I+ eluate was passed through a (sterile) 0.2 micron filter. filtered and then frozen.

Immediately after washing the column as previously described, 0.02M sodium citrate, pi (from 0.05M NaCl in 6,8 to 0.6M NaCl, 150 liter of linear salt gradient at a flow rate of 650 m1/min. applied to the system. A 3.25 liter portion of the column eluate was added to the gradient. and each third fraction was measured for its factor IX and X activity. It was tested in order to After completion of this gradient, 0.02M sodium citrate , an additional 75 liters of solution containing 0.5M NaCl in pH 6.8. this applied to the column and collected a 3.25 liter portion of this eluate, and [Factor] X and the activity of X were assayed. Contains relatively high factor X activity but low factor The fractions containing factor X activity were pooled to create a factor X eluate pool. second factor The eluate pool was transferred to a dye using a Millipore Pelicon concentrator. Concentrated by filtration. After concentration, this factor X eluate was (sterilized) Filtered through a 0.2 micron filter and then frozen.

Factor 11-, IX-, X-containing materials from the starting concentrate and the above enriched factor I] Boules were assayed for factor 11 activity and protein content. factor] [-1■-1I The X- and X-containing concentrates and the pools of concentrated Factor Assayed for protein content. The results of these assays are shown in Table 1.

Total units Yield 2 Specific activity Plasma fraction 1.4 x 10 @ book -4,79 DSS a degree' 8.91 x 1 0' 63.6% 5.38 factor Plasma fraction 6.3 x 10' book-2,2DSS a degree 6.67 x 10' 10.6% 20.5 factor V Nemoto Plasma fraction ND O,017 Ba supernatant ND 46% obtained from a separate preparation of 0.027 Ne45, Estimates from the average factor 1 [/factor IX ratio and the average factor X/factor IX ratio.

This book: Results for factor ■ based on another laboratory experiment using other methods.

No data could be obtained from the preparation described in Example I.

ND Not measured.

1. Concentrated eluate from the first step of dextran sulfate chromatography.

2% yield is the number of units recovered in the above steps compared to the number of units in the starting material. The number of units obtained is multiplied by 100.

The purity (specific activity) of the factor [() was determined by dextran sulfate chromatography to 1 It increased by 2%. The purity of Factor V was increased by 60% by barium precipitation. of factor Purity was increased 10 times by dextran sulfate chromatography.

Approximately 460 mg of Factor Sali snake venom and 5 mg (aclt) in a total volume of 1.300 mg at 37 °C. and incubate for 30 minutes to convert Factor X to Factor X. This activation At the end of the process, the snake venom was transferred to a column chromatogram on benzamidine-Sepharose. It was removed by matography. This benzamidine-sepharose was added to 0.0 2 breasts! Equilibration was carried out with HEPES, pH 7, 4, 0, 15M NaCl.

This activated factor and combined them. The unbound substance was mixed with 0.02M HEPES, pH 7.4, The above chromatography was performed using 0,15M NaCl, 0,005M benzamidine. eluted from the feed medium. This eluted factor X was collected and 0.02M T ris, pHs, s, 0.15 M dialyzed against NaCl, and 0.1 (wt/wt)% albumin and 1.0 (wt/wt)% polyethylene glyco It was stabilized by the addition of alcohol.

Activation and purification of L. Brought.

Example 3 Mix 75mg of phosphatidylserine and 225mg of phosphatidylcholine and sonicate for 30-45 min at 4 °C to create a synthetic phospholipid film. Ta.

200 mg of Factor 1.1 prepared according to the procedure of Example 1 was added to 0.0 at pH 7.3 containing 0.09 M NaCl and 4 μg of factor , 06M Tris buffer, diluted to 5 mg/ml, containing factor V. A 0 μm barium chloride supernatant was prepared according to the procedure of Example 1: IOn moles/ml. Phospholipids were prepared according to the procedure of Example 3 +7 mM CaC1z, 0.3 (w t/wt)% Tri-(n)butyl phosphate and 1(Wt/Wt)% Polysorbate 80 was added. The reaction mixture was incubated at room temperature for 6 hours. It was incubated.

At the end of the incubation, the pH of this mixture was adjusted to 6.7 and P Addition of EG brings the final concentration of 5 (Wt/Wt)% PEG and The mixture was incubated for 30 minutes at room temperature. 3. This precipitate. At OOOrpm The resulting PEG supernatant, removed by centrifugation for 30 min, was diluted with an equal volume of distilled water. and 9.2 ml sulfapropyl-Sephadex (6,5 p 0,0E in H) and 1 Mixed at 4°C for an hour. This factor 11-containing sulfapropyl-sephadec The column was then rinsed with 0.01M sodium citrate at a pH of 6.7. Washed with thorium. This factor II was then treated with 0.1 M quench at a pH of 6°5. It was eluted with sodium chloride. The specific activity of 1.220 units/A0° unit is 85 obtained by a small-scale activation procedure with a yield of %. The results are summarized in Table II. Ru.

! Dan Total unit yield book specific activity (units) % (units Reaction mixture 157. θ00 100 560 PEG supernatant 130.000 83.500 zero% yield is calculated at the above stage (this) compared to the number of units in the starting material. 100 times the number of units collected.

Mind Pyrocut scale activity from factor II to factor ratio was prepared according to the procedure of Example 1. 20 g of Factor II was added to a solution containing 0.09 M NaCl prepared according to Procedure C2 of Example 2. 0,06M Tris at pH 7.3 (5mg/ml in -7 fur) 4 mg of Factor X was prepared according to the sum of Example 2.

100ml chloride/<1Noum supernatant containing factor ■ prepared according to the procedure of Example 1 , and this solution was brought to the final concentration (1) and then followed the procedure of Example 3. 28 nmol/ml of 100% lactose, 7 mM CaC12,0 prepared according to , 3(wt/wt)% Tri-(n)butyl phosphate and 1(wt/w t)% polysorbate 80 was added. The reaction mixture was brought to room temperature) for 6 hours. Incubated for hours. ps of this mixture at the end of the incubation. The pH was adjusted to 6.5 and 5 (wt/wt)% PEG was added by addition of solid PEG. and incubated at room temperature for 30 minutes to remove the PEG. Dissolved. This mixture was then heated for 30 minutes at 3.000 rpm. The hearts were separated and the PEG precipitate was removed. The obtained PEG supernatant was added to an equal volume of Dilute with distilled water and add 11 sulfapropyl-Sephadex (6,5 p 0 in H, O1^(sodium citrate (which is equilibrated from this) and mixed for 1 hour at 4°C. This factor II-containing sulfapropyrusef Addex was then added to the column (two rinses and a 0.01M solution at a pH of 6.5). Washed with sodium enoate. This factor It, then 0 at pt+ of 6.7 , eluted with 1M sodium citrate. 1. Specific activity of 100 units/ml , obtained by a pilot scale activation procedure with a yield of 75%. This result is shown in the table below. In III (required total units, yield, specific activity) (units) % (units Activation mixture 21 100 520 PEG supernatant 17.9 85.2 480SP-7) T de, x Eluate 15.7 74.8 1.100 % yield is in units of starting material The number of units recovered in the above step compared to the number multiplied by 100.

Factor I! , v, rx and X for purification, and factor X of factor X.

The above description of exemplary embodiments for activation to belongs to. As modifications will be apparent to those skilled in the art, the present invention is not limited to the foregoing description. It is not intended to be limited to the particular embodiments described. Also, the disclosed invention is performed in the absence of any elements not specifically disclosed herein. I can do two things. The scope of the invention is defined by the following claims.

Continuation of front page (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , 0A (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN.

TD, TG), AT, AU, BB, BG, BR, CA.

CH, CZ, DE, DK, ES, FI, GB, HU, JP, KP , KR, LK, LU, MG, MN, MW, NL, NO, NZ, PL , PT, RO, RU, SD, SE.

SK, U.A. (72) Inventor: Unimura, Hahiro United States, California 91006° Arcadia, South Sun Ta Anita Avenue 408. #12 (72) Inventor Munehiro Noda 515 Tamachi, Tenri City, Nara Prefecture (72) Inventor Shitaniji, Kenneth Tee.

United States, California 91607°F. hollywood, simp Son Avenue 5501

Claims (26)

[Claims] 1. Factor II, Factor V from a single impure protein fraction containing Factors II, V and X and Factor X, each of which comprises: contacting the aqueous solution with the first ligand, thereby causing the factor II, V and X are bound to the first ligand; from this first ligand factor I Collect factor I, V and X protein fractions; barium chloride to the X protein fraction to form a barium precipitate comprising Factor II and X; Recover factor V from the supernatant of the barium precipitate; Dissolving to make a factor II- and factor X-containing solution; This factor II- and factor X-containing solution is capable of binding factor applied to a second ligand that binds weakly or not at all to II; Factor X binds to the second ligand; does not bind to the second ligand; or recovering factor II from the weakly bound fraction; and Recovering Factor X from the second ligand. 2. 2. The method of claim 1, wherein the first ligand is a DEAE ligand. 3. Factor II, V and X protein fractions were eluted with approximately 0.2M NaCl to DE 3. The method of claim 2, wherein the method is recovered from AE ligand. 4. Factor II, V and X protein fractions to a final concentration of about 0.01M to about 0.22M 2. The method according to claim 1, wherein the precipitation is performed by adding barium chloride. 5. 2. The method of claim 1, wherein the second ligand is dextran sulfate. 6. Recovering factor X from the second ligand by elution with 0.2M NaCl; The method according to claim 1. 7. A method for separating factor II from impure protein fractions containing factor II. So; Prepare an aqueous solution of the impure protein fraction described above; contact this aqueous solution with a first ligand. to cause Factors II, V and X to bind to the first ligand; Collect factor II, V and X protein fractions from the first ligand; Add to the collected factor II, V and make a barium precipitate; dissolve this barium precipitate in an aqueous solution, factor II- and Making a factor X-containing solution; This factor II- and factor X-containing solution is capable of binding factor applied to a second ligand that binds weakly or not at all to II; and Factor II is recovered from the fraction that does not bind or binds weakly to this second ligand. Ru, A method comprising: 8. 8. The method of claim 7, wherein the first ligand is a DEAE ligand. 9. Factor II, V and X protein fractions were eluted with approximately 0.2M NaCl to DE 9. The method of claim 8, wherein the method is recovered from AE ligand. 10. Salt the Factor II, V and X protein fractions to a final concentration of about 0.1M to about 0.2M. 8. The method according to claim 7, wherein the precipitation is carried out by adding barium chloride. 11. 8. The method of claim 7, wherein the second ligand is dextran sulfate. 12. A method for separating factor V from impure protein fractions containing factor V. hand; Prepare an aqueous solution of the impure protein fraction described above; contact this aqueous solution with a first ligand. to cause Factors II, V and X to bind to the first ligand; Collect factor II, V and X protein fractions from the first ligand; Add to the collected factor II, V and make a barium precipitate; and recover factor V from the supernatant of this barium precipitate. , a method comprising: 13. 13. The method of claim 12, wherein the first ligand is a DEAE ligand. 14. Factor II, V, and X protein fractions were eluted with approximately 0.2 M NaCl. 14. The method of claim 13, wherein the method is recovered from an EAE ligand. 15. Salt the Factor II, V and X protein fractions to a final concentration of about 0.1M to about 0.2M. 13. The method according to claim 12, wherein the precipitation is carried out by adding barium chloride. 16. 1. A method for activating factor II to factor IIa, comprising: an activation buffer; - factor II [herein, factor II is composed of the following; factors II, V and X; preparing an aqueous solution of an impure protein fraction; This aqueous solution is contacted with a first ligand, and Factors II, V and X are added to the first ligand. combined with; Factor II, V and X protein fractions were recovered from this first ligand; A buffer comprising factors II and X is added to the recovered factor II, V and X protein fractions. make a barium precipitate; dissolve this barium precipitate in an aqueous solution, factor II- and make a factor X-containing solution; This factor II- and factor X-containing solution is combined with factor X but weakly with factor II. to a second ligand that binds or does not bind at all; and remained unbound or weakly bound to this factor Recovering Factor II from the fraction. ] diluted and providing a Factor II solution; adding Factor V to the Factor II solution to provide a Factor II/Factor V solution; Factor Xa [here, factor Xa; Recovering Factor X from the second ligand of the Factor II preparation procedure described above; and is prepared by activating this factor X by specific proteolytic cleavage. . ] to this Factor II/Factor V solution to form a Factor II/Factor V/Factor Xa solution. provide the phospholipid membrane and calcium ions to this Factor II/Factor V/Factor added to the Xa solution; and incubating the resulting mixture to activate Factor II to Factor IIa; A method comprising: 17. 17. The method of claim 16, wherein the first ligand is a DEAE ligand. 18. Factor II, V, and X protein fractions were eluted with approximately 0.2 M NaCl. 18. The method of claim 17, wherein the method is recovered from an EAE ligand. 19. Salt the Factor II, V and X protein fractions to a final concentration of about 0.1M to about 0.2M. 17. The method according to claim 16, wherein the precipitation is carried out by adding barium chloride. 20. 17. The method of claim 16, wherein the second ligand is dextran sulfate. 21. Factor X is recovered from the second ligand by elution with approximately 0.2M NaCl. 17. The method of claim 16. 22. Factor X was added to Russell's viper venom in the presence of calcium ions ell's viper venom). 17. The method according to claim 16, wherein the method is further activated. 23. Recovering Factor V from the barium supernatant produced by the Factor II preparation procedure; 17. The method according to claim 16. 24. Claim 1, wherein Factor V is provided by a portion of plasma or cryoprecipitated plasma. 6. The method described in 6. 25. 17. The concentration of phospholipids is from about 5 to about 50 nmol/ml. Method. 26. 17. The method of claim 16, wherein the calcium level is about 2 to about 20 mM.